EP2621539A1 - Nanoparticules auto-assemblantes pour la libération de bisphosphonates dans le traitement des cancers humains - Google Patents

Nanoparticules auto-assemblantes pour la libération de bisphosphonates dans le traitement des cancers humains

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Publication number
EP2621539A1
EP2621539A1 EP11776382.1A EP11776382A EP2621539A1 EP 2621539 A1 EP2621539 A1 EP 2621539A1 EP 11776382 A EP11776382 A EP 11776382A EP 2621539 A1 EP2621539 A1 EP 2621539A1
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Prior art keywords
nanovectors
lipid
nps
ligand
zol
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German (de)
English (en)
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EP2621539B1 (fr
Inventor
Giuseppina Salzano
Monica Marra
Carlo Leonetti
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ABBRUZZESE SACCARDI, ALBERTO
CARAGLIA, MICHELE
DE ROSA, GIUSEPPE
TASSONE, PIERFRANCESCO
Istituti Fisioterapici Ospitalieri IFO
Original Assignee
Caraglia Michele
De Rosa Giuseppe
La Rotonda Maria Immacolata
ROTONDA MARIA IMMACOLATA
Istituti Fisioterapici Ospitalieri IFO
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention refers to the field of pharmaceutical formulations containing bisphosphonates for the treatment and prevention of human cancers.
  • BPs Bisphosphonate
  • NBPs amino-BPs
  • ZOL zoledronic acid
  • BPs induce apoptosis of tumour cells and inhibit tumour cell growth, in vitro, of a variety of tumour cell types, including breast (Senaratne et al. 2000, Jagdev et al. 2001 a), prostate (Lee et al. 2001 ), melanoma (Riebeling et al. 2002), osteosarcoma (Mackie et al. 2001 , Sonnemann et al. 2001 ) and myeloma (Shipman et al. 1998) tumour cells.
  • ZOL belongs to the class of NBPs that are used for the treatment of the complications derived from bone metastases so called skeletal related events (pain, spontaneous fractures, bone radiotherapy requirement etc.) and for the prevention of the cancer treatment induced osteopenia (hormone therapy in the treatment of prostate and breast cancer).
  • skeletal related events pain, spontaneous fractures, bone radiotherapy requirement etc.
  • cancer treatment induced osteopenia hormone therapy in the treatment of prostate and breast cancer.
  • up-to-date ZOL has not still been used as a drug with direct anti-cancer activity and its ability to act directly against bone metastases has never been demonstrated also for the limits of radiologic detection and measurement of cancer in the bone.
  • no survival advantage was reported in the patients treated with NBP if compared with patients not treated with NBP, with the exception of the study by M. Gnant et al (N Engl J Med. 2009) that demonstrates a survival advantage in the adjuvant setting of hormone-dependent breast cancer treated with ZOL.
  • the formulation strategy developed so far present some inconvenient such as a reduced liposomes physical stability which requires the liposomes to be lyophilised, a low encapsulation efficiency (about 5% corresponding to 100 ⁇ g ZOL/mg of lipids) and a certain drug loss following rehydratation after lyophilization.
  • WO2008/005509 in which BPs are used as hydroxyapatite binding agents.
  • the invention describes particles in which BP is used to bind biological surface, such as tooth or bone.
  • the patent application WO2008/005509 describes PLGA-PEG nanoparticles chemically conjugated at the carboxyl terminal to a biphosphonate which were added in trace amount to a solution of CaCO3 (average size 800nm) resulting in an aggregation of the CaCO3 particles.
  • Such a composition is suitable for delivering particles to a tooth or bone.
  • this composition can be used to modify biological surface of tooth or bone, while is not suitable to avoid BP accumulation into the bone and cannot be used to target tumors.
  • the object of the present invention are nanocomplexes, also called nanoparticles (NP), containing bisphosphonates complexed with inorganic and lipid nanovectors; wherein the biphosphonate is first complexed to inorganic nanovectors and the resulting particles are then complexed to lipid nanovectors and wherein the inorganic nanovectors consist of nanoparticles of a Ca-, Mg-, Sr- or Zn- inorganic salt.
  • the aforementioned nanocomplexes can be also named self-assembling nanoparticles.
  • the aforementioned nanocomplexes are useful as pharmaceutical formulation for the treatment or the prevention of tumor growth and/or metastasis.
  • the tumors can be solid or haematological tumors, such as prostate cancer, lung cancer, head/neck cancer, colon cancer, liver cancer, breast cancer, pancreas cancer, kidney cancer, bladder cancer, male and female urogenital tract cancer, bone cancer, multiple myeloma, melanoma, lymphoma, primitive and secondary tumors.
  • solid or haematological tumors such as prostate cancer, lung cancer, head/neck cancer, colon cancer, liver cancer, breast cancer, pancreas cancer, kidney cancer, bladder cancer, male and female urogenital tract cancer, bone cancer, multiple myeloma, melanoma, lymphoma, primitive and secondary tumors.
  • the invention related to a bisphosphonate, for example the zoledronic acid (ZOL), complexed with nanoparticles based on calcium and phosphate salts; such particles are mixed with other particles, for example liposomes.
  • ZOL zoledronic acid
  • the nanocomplexes, object of this invention have advantages such as the possibility to be easily prepared immediately before use, a high drug loading, a high reproducibility of the results.
  • Nanocomplexes, according with this invention are nanoparticulate entities, for which, surprisingly, imagines were acquired by cold field electron gun scanning electron microscopy (cFEG-SEM) (figure 1 ). These nanocomplexes have a mean diameter ranging form 10 to 500 nm. Moreover, such system has showed an antitumor effect higher than that observed with ZOL- containing liposomes previously developed (Marra et al. Biotechnology Advances 201 1 ).
  • Figure 1 It shows images of cFEG-SEM, acquired with a magnification of
  • Figure 2 Example of luminescence associated to injected tumour cells in an untreated mouse (upper panel) and in a mouse treated with nanocomplex according to the invention (PLCaPZ NPs;lower panel) achieving a complete regression of the tumour after 56 days from the initial tumour cell injection.
  • bisphosphonates, in pharmaceutical formulation based on the combination of lipid and inorganic nanovectors can be successfully used for the treatment of different solid and haematological tumors, such as prostate cancer, lung cancer, head/neck cancer, colon cancer, liver cancer, breast cancer, pancreas cancer, kidney cancer, bladder cancer, male and female urogenital tract cancer, bone cancer, multiple myeloma, melanoma, lymphomas.
  • ligands can be added on the NP surface in order to target specific cells.
  • Ligands can be selected among those having specificity for receptors that are over-expressed on specific cells, for example cancerous cells, but are normally or minimally expressed on normal, healthy cells. These molecules should have high affinity to their cognate receptors, plus can have innate abilities to induce receptor-mediated endocytosis.
  • the targeting layer poses as the outmost exterior of the NP, where targeting ligands are generally presented on top of the stealth layer. Structures such as antibodies, antibody fragments, proteins, small molecules, aptamers and peptides have all demonstrated abilities to induce NP-targeting to cancer cells (M. Wang and M. Thanou , Pharmacological Research 2010; Huynh,et al. Nanomedicine 2010.).
  • NP with human-transferrin on their surface were prepared and characterized (see experimental section).
  • the Formulation can be administered to the patient by parenteral administration, for example by intravenous, intraperitoneal, intratumoral, intrarterial injection, depending on the type of tumor to treat.
  • parenteral administration for example by intravenous, intraperitoneal, intratumoral, intrarterial injection, depending on the type of tumor to treat.
  • Other routes of administration such as oral or trasdermal, cannot be excluded.
  • the formulation can be used for different BPs, such as clodronate, alendronate, etidronate, pamidronate, tiludronate, ibandronato, neridronato, zoledronate, minodronate and risedronate, and their biological derivates or prodrugs.
  • the selected BP is preferably the zoledronic acid.
  • the amount of BP loaded into the nanoparticles is preferably comprised between 0.001 ⁇ g e 100 mg di BP/ml of suspension, more preferably between 50 ⁇ g and 0.250 mg BP/ml of suspension.
  • the nanovectors are structures with a size ranging between ten and hundreds of nanometers able, when associated to a drug, to change its pharmacokinetic profile.
  • the nanovectors are nanoparticles, nanocapsules or nanospheres, liposomes or other nanovectors such as niosomes, micelles.
  • the nanovectors have to be preferably prepared by mixing of solutions and/or dispersions.
  • the formulation consists in self-assembling nanovectors prepared before use.
  • the nanovectors can be prepared by mixing two or more aqueous or organic solutions or suspensions.
  • the nanovectors can be lipid or polymeric nanoparticles, for example liposomes or other nanovectors such as niosomes and micelles.
  • nanovectors are preferably self-assembling nanoparticles, preferably stealth nanoparticles (NPs bearing an hydrophilic polymer, i.e. polyethylene glycol or PEG, on their surface).
  • NPs bearing an hydrophilic polymer i.e. polyethylene glycol or PEG, on their surface.
  • the self-assembling nanoparticles are preferably formed starting from inorganic nanoparticles and lipid nanovectors.
  • the inorganic nanovectors are nanoparticles based on inorganic salts.
  • inorganic salts containing Ca, Mg, Sr, Zn, and mixtures thereof.
  • these inorganic nanovectors are based on Ca and P and, in particular, on nanoprecipitated calcium hydrogen phosphate.
  • the lipid nanovectors are preferably liposomes, preferably composed on phosphoglycerides and sfingolipids, together with their products of hydrolysis, sterols, cationic lipids, anionic lipids, neutral lipids, lipids conjugated with synthetic or natural polymers or lipids bound to fluorescent probes, or lipids bound to proteins or peptides, or lipids conjugated with molecules able to specifically interact with receptor of the cell membrane.
  • these liposomes are based on mix of 1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), cholesterol, 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), preferably in amount ranging between 0-100%, 0-80% and 100-0% (p/p), respectively.
  • DOTAP 1 ,2-dioleoyl-3-trimethylammonium-propane
  • DSPE-PEG 2000 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]
  • DSPE-PEG 2000 is premixed with the other components of the liposomal mix.
  • the invention concerns to a method for the preparation of the complexes, comprising the following steps:
  • a suspension of the lipid nanovectors with the suspension obtained from the step a.
  • the ligand can be added, incubating the NP obtained according to the process as above described, in a solution containing the ligand; or, in alternative, it can be included in the NP first incubating the lipid nanovectors in a solution containing the ligand and then mixing the obtained ligand-nanovectors complexes or conjugated with the suspension obtained from the step a.
  • the invention concerns a kit to prepare the aforementioned pharmaceutical formulations; such kit contains:
  • the kit according to the invention can include at least a container containing a ligand, or a solution thereof, for receptors overespressed in cancer cells.
  • the ligand can be already present into the container containing a suspension containing lipid nanovectors.
  • Example 1 The present invention can be better understood from the following examples.
  • Example 1
  • Step 1 Preparation of calcium phosphate nanoparticles and their complexes with ZOL
  • Liposomes consisting of DOTAP/chol (1 :1 weight ratio) or DOTAP/chol/DSPE- PEG 2000 (1 :1 :0.5 or 1 :1 :1 weight ratio) were prepared by hydration of a thin lipid film followed by extrusion. Briefly, the lipid mixture were dissolved in 1 ml of a mixture chloroform/methanol (2:1 v/v), the resulting solution was added to a 50 ml round-bottom flask, and the solvent was removed under reduced pressure by a rotary evaporator under nitrogen atmosphere.
  • the lipid film was hydrated with 1 ml of sterile water and the resulting suspension was gently mixed in the presence of glass beads, after that the flask was left at room temperature for still 2 h.
  • the liposome suspension was then extruded using a thermobarrel extruder system passing repeatedly the suspension under nitrogen through polycarbonate membranes with decreasing pore sizes (0.4-0.1 ⁇ ). After preparation, liposomes were stored at 4°C. Each formulation was prepared in triplicate.
  • Step 3 Preparation of self assembly nanoparticles containing ZOL
  • CaP ZOL NP obtained according to Step 1 were mixed with cationic liposomes obtained according to Step 2. Briefly, 500 ⁇ of CaPZ NPs were mixed with 500 ⁇ of DOTAP/chol liposomes, at a final ZOL concentration 0.25 or 0.05 mg/ml suspension. One milliliter of a LCaPZ NPs suspension was then mixed with 50 ⁇ micellar dispersion of DSPE-PEG2000 (47 mg/ml) and then incubated at 50-60 °C for 10 min. The resulting suspension (post-PLCaPZ NPs) was then allowed to cool to room temperature before use.
  • Step 1 and Step 2 was similar to that described in Example 1 .
  • Step 3 Equal volumes of suspensions containing DOTAP/chol/DSPEG2000 liposomes and CaPZ NPs, respectively, were mixed in a glass tube and the resulting dispersion was allowed to stand at room temperature for 10 min (pre- PLCaPZ NPs).
  • the mean diameter of cationic liposomes and PLCaPZ NPs were determined at 20 °C by photon correlation spectroscopy (PCS). Each sample was diluted in deionizer/filtered water and analyzed with detector at 90° angle. As measure of the particle size distribution, polydispersity index (P.I) was used. For each batch, mean diameter and size distribution were the mean of three measures. For each formulation, the mean diameter and P.I. were calculated as the mean of three different batches.
  • zeta-potential ( ⁇ ) of the NPs surface was measured in water by means of a Zetasizer Nano Z. Data of ⁇ were collected as the average of 20 measurements.
  • Morphological analysis of LCaPZ, post- and pre-PLCaPZ NPs were investigated by cold field emission gun-scanning electron microscopy (cFEG-SEM), as reported by De Rosa et al. (De Rosa et al. International Journal of Pharmaceutics 2008). For cFEG-SEM analysis, samples were prefixed in a mix of 4% formaldehyde and 1 % glutaraldehyde in distilled water for 1 h.
  • samples were rinsed in distilled water by ultracentrifugation (80,000 rpm) and post-fixed by adding 1 % OsO4 for 1 h. After a further washing with distilled water, pellets were filtered on a polycarbonate filter (0.1 ⁇ ) in a Swinnex filtration apparatus (Millipore, USA). A second filter was placed over the first to form a sandwich in which liposomal pellets were trapped. Samples were then dehydrated in a graded alcohol series (10, 30 and 50% for 10min, 70 and 80% for 30min, 95% for 1 h and 100% overnight at 4 °C) and critically point dried.
  • a graded alcohol series (10, 30 and 50% for 10min, 70 and 80% for 30min, 95% for 1 h and 100% overnight at 4 °C) and critically point dried.
  • the Swinnex was opened and both filters were placed on a stub cleaned with acetone to remove any grease.
  • Double adhesive carbon disks EMS, USA
  • EMS Double adhesive carbon disks
  • the filters containing the samples were placed over it.
  • the stubs were sputter coated with a nanometric layer of gold.
  • Observations were carried out by a cold cathode Field Emission Gun Scanning Electron Microscope (FEG Jeol 6700F, Jeol Ltd., Japan). The pictures of the NPs surface at higher magnifications were taken at 2-5 kV (see Figure 1 ).
  • ZOL analysis was carried out by reverse phase high performance liquid chromatography (RP-HPLC).
  • the HPLC system consisted of an isocratic pump equipped with a 7725i injection valve, SPV-10A UV-Vis detector set at the wavelength of 220 nm.
  • the system was controlled by a SCL-10A VP System Controller connected with a computer.
  • Chromatograms were acquired and analysed by a Class VP Client/Server 7.2.1 program.
  • the quantitative analysis of ZOL was performed on a Gemini 5 ⁇ C18 column (250 X 4.60 mm, 1 10 A Phenomenex, Klwid, USA) equipped with a security guard.
  • the mobile phase was a mixture 20:80 (v/v) of acetonitrile and an aqueous solution (8 imM di-potassium hydrogen orthophosphate, 2 imM di-sodium hydrogen orthophosphate and 7 imM tetra-n-butyl ammonium hydrogen sulphate, adjusted at pH of 7.0 with sodium hydroxide).
  • ZOL determination was performed in isocratic condition, at a flow rate of 1 ml/min at room temperature.
  • the amount of un-complexed ZOL was determined as follows: 1 ml of NPs dispersion was ultracentifugated (Optima Max E, Beckman Coulter, USA) at 80.000 rpm at 4°C for 40 min. Supernatants were carefully removed and ZOL concentration was determined by RP-HPLC. The results have been expressed as complexation efficiency, calculated as the ratio between the amount of ZOL present in the supernatants and the amount of ZOL theoretical loaded.
  • CD-1 nude (nu/nu) mice 6-8 weeks old were purchased from Charles River Laboratories (Milano, Italy).
  • mice have been acclimated for one week before to be injected with tumor cells. Mice were injected intramuscularly with tumor cells at 3 x 106 cells/mouse in 200 ⁇ of PBS. Six days after, a tumor mass of about 0.3 cm3 in diameter was evident, mice were randomized in four groups. Six mice/group were treated with ZOL free or NP containing ZOL for 3 consecutive weeks. Tumor mass was calculated three times a week from caliper measurements by using the formula ⁇ /6 AB, where A is the major diameter and B is the minor diameter, corresponding to tumor mass (Sommer K. et al. International Journal of Oncology 2001 ). Toxicity was evaluating by measuring the body weight two times a week. Student's t test was used for statistical comparison of differences.
  • PC3M-luc2 is a luciferase expressing cell line which was stably transfected with the Iuc2 firefly luciferase gene (Caliper Life Sciences, Hopkinton, MA, USA). Human xenografts were imaged using the IVIS imaging system 200 series (Caliper Life Sciences). Briefly, mice were anesthetized with a combination of tiletamine- zolazepam (Telazol, Virbac, Carros, France) and xylazine (xylazine/ Rompun BAYER) given intramuscularly at 2 mg/kg.
  • tiletamine- zolazepam Telazol, Virbac, Carros, France
  • xylazine xylazine/ Rompun BAYER
  • mice were injected intraperitoneal ⁇ with 150 mg/kg D-luciferin (Caliper Life Sciences), and imaged in the supine position 10-15 min after luciferin injection. Data were acquired and analyzed using the living image software version 3.0 (Caliper Life Sciences).
  • TUNEL assay after washing in PBS supplemented in 0.1 % BSA, cells were treated with an in situ detection kit, according to the manufacturers instructions (Boehringer Mannheim Biochemicals). Nuclei with fragmented DNA were visualized by a fluorescence microscope.
  • pre-PLCaPZ NP with a mean diameter of about 150 nm with Pl ⁇ 0.2.
  • the actual loading of ZOL in pre-PLCaPZ NP was of about 0.175 mg/ml of dispersion, corresponding of a complexation efficiency of about 70%.
  • the amount of ZOL loaded in post PLCaPZ NP was 5 times lower than that found in pre PLCaPZ NP, since an increase in concentration of ZOL from 0.05 to 0.25 results in aggregates formation.
  • Table 1 Diameter, P. I., zeta potential ( ⁇ ) and complexation efficiency of post and pre-PLCaPZ NPs.
  • Figure 1. Images from cFEG SEM analysis.
  • A LCaPZ NPs;
  • B Pre-PLCaPZ NPs;
  • C Post-PLCaPZ NPs. Scale bar 100 nm.
  • pre-PLCaPZ NPs caused an about 12-fold potentiation of ZOL-induced cytotoxicity, while a lower effect (about 8-fold) was found with post-PLCaP NPs.
  • PC-3 cells were injected into the left hind leg muscles of nude mice and six days after (when a tumor mass was evident) treatment started by intravenously injection of 20 ⁇ g of ZOL free or complexed with pre PLCaPZ NP, a formulation selected for the best technological characteristics.
  • NP pre PLCaPZ NP
  • a group of mice was treated with NP not containing ZOL, to evaluate the potential antitumoral effects. Mice were treated three times a week for three consecutive weeks.
  • T and C are the median times for treated (T) and untreated tumors (C), respectively, to achieve equivalent size.
  • Step 1 Preparation of PEGylated cationic liposomes
  • Liposomes consisting of DOTAP/chol/DSPE-PEG 2000 (1 :1 :0.5 weight ratio) were prepared by hydration of a thin lipid film followed by extrusion. Briefly, the lipid mixture were dissolved in 1 ml of a mixture chloroform/methanol (2:1 v/v), the resulting solution was added to a 50 ml round-bottom flask, and the solvent was removed under reduced pressure by a rotary evaporator under nitrogen atmosphere. Then, the lipid film was hydrated with 1 ml of sterile water and the resulting suspension was gently mixed in the presence of glass beads, after that the flask was left at room temperature for still 2 h.
  • the liposome suspension was then extruded using a thermobarrel extruder system passing repeatedly the suspension under nitrogen through polycarbonate membranes with decreasing pore sizes (1 .4-0.4 ⁇ ). After preparation, liposomes were stored at 4°C. Each formulation was prepared in triplicate.
  • Step 2 Preparation of calcium phosphate nanoparticles and their complexes with ZOL
  • CaP Z NPs CaP/ZOL-NPs complexes
  • Step 3 Preparation of self-assembly NPs containing ZOL
  • Step 4 Preparation of post-Tf-PLCaPZ NPs
  • PLCaPZ NPs bearing Tf on their surface were prepared by incubation of PLCaPZ NPs dispersion, prepared according to Step 3, with Tf solutions (10 mg/ml in phosphate buffer at pH 7.4) for 15 min, at room temperature, at a volume ratio of 1 :0.5.
  • Step 1 Preparation of Transferrin-PEGylated cationic liposomes
  • Tf-PEGylated cationic liposomes were prepared by pre-incubating of PEGylated cationic liposomes, obtained according to Step 1 of the Example 3, with human transferrin (10 mg/ml in phosphate buffer at pH 7.4) at a volume ratio of 1 :1 . The resulting liposomes were allowed to stand at room temperature for 15 min before the use.
  • Step 2 Preparation of pre-Tf-PLCaPZ NPs
  • Tf-PEGylated cationic liposomes prepared according to Step 1 , were mixing with CaPZ NPs, prepared according to Step 2 of the Example 3, at a volume ratio of 1 :0.5. The resulting dispersion was incubated at room temperature for 15 min before the use.
  • Tf-PLCaPZ NPs The mean diameter of Tf-PLCaPZ NPs, were determined at 20°C by photon correlation spectroscopy (PCS) (N5, Beckman Coulter, Miami, USA). Each sample was diluted in deionizer/filtered (0,22 ⁇ pore size, polycarbonate filters, MF- Millipore, Microglass Heim, Italy) water and analyzed with detector at 90° angle. As measure of the particle size distribution, polydispersity index (P.I) was used. For each batch, mean diameter and size distribution were the mean of three measures. For each formulation, the mean diameter and P.I. were calculated as the mean of three different batches.
  • PCS photon correlation spectroscopy
  • zeta-potential ( ⁇ ) of the NPs surface was measured in water by means of a Zetasizer Nano Z (Malvern, UK). Data of ⁇ were collected as the average of 20 measurements.
  • ZOL analysis was carried out by reverse phase high performance liquid chromatography (RP-HPLC).
  • RP-HPLC reverse phase high performance liquid chromatography
  • the amount of un-complexed ZOL was determined as follows: 1 ml of NPs dispersion was ultracentifugated (Optima Max E, Beckman Coulter, USA) at 80.000 rpm at 4°C for 40 min. Supernatants were carefully removed and ZOL concentration was determined by RP-HPLC. The results have been expressed as complexation efficiency, calculated as the ratio between the amount of ZOL present in the supernatants and the amount of ZOL theoretical loaded. Characteristic of pre and post-Tf-PLCaPZ NPs

Abstract

La présente invention concerne des nanocomplexes également appelés nanoparticules auto-assemblantes comprenant des biphosphonates, des nanovecteurs lipidiques et des nanovecteurs inorganiques. L'invention concerne en particulier de l'acide zolédronique complexé avec des nanoparticules à base de phosphate de calcium; lesdites particules sont à leur tour mélangées à des particules lipidiques, par exemple des liposomes. Lesdits nanocomplexes sont utiles et se sont révélés efficaces in vivo comme préparations pharmaceutiques de biphosphonates pour le traitement ou la prévention de la croissance tumorale et/ou des métastases. Les tumeurs peuvent être solides et/ou hématologiques, comme dans le cas de la prostate, du poumon, de la tête/du cou, du colon, du foie, du sein, du pancréas, des reins, de la vessie, du tractus urogénital, des os, des myélomes multiples, des tumeurs primitives et secondaires du système neveux central et des lymphomes.
EP11776382.1A 2010-10-01 2011-09-30 Nanoparticules auto-assemblantes pour la libération de bisphosphonates dans le traitement des cancers humains Active EP2621539B1 (fr)

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ITFI2010A000206A IT1401882B1 (it) 2010-10-01 2010-10-01 Nanoparticelle autoassemblanti per il rilascio di bifosfonati nel trattamento di tumori.
PCT/EP2011/067119 WO2012042024A1 (fr) 2010-10-01 2011-09-30 Nanoparticules auto-assemblantes pour la libération de bisphosphonates dans le traitement des cancers humains

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EP3494974B1 (fr) 2011-07-08 2023-10-18 The University of North Carolina at Chapel Hill Nanoparticules de métal-bisphosphonate pour thérapie anticancéreuse et imagerie, ainsi que pour traiter des troubles des os
RU2519120C1 (ru) * 2012-11-06 2014-06-10 Федеральное государственное бюджетное учреждение "Ростовский научно-исследовательский онкологический институт" Министерства здравоохранения Российской Федерации Способ профилактики скелетных осложнений у больных с литическими метастазами в плоские и смешанные кости
GB201319363D0 (en) 2013-11-01 2013-12-18 Uni I Oslo Compounds
JP6590802B2 (ja) 2013-11-06 2019-10-16 ザ ユニバーシティ オブ シカゴＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃｈｉｃａｇｏ 化学療法用薬剤、核酸及び光増感剤の送達又は共送達のためのナノスケール輸送体
KR101577271B1 (ko) 2014-03-24 2015-12-15 가톨릭대학교 산학협력단 칼슘 이온 및 친수성 비스포스포네이트를 이용한 나노복합체의 제조방법 및 상기 나노복합체를 이용한 약물전달체
EP3377043A4 (fr) * 2015-11-20 2019-06-26 The Regents of The University of California Véhicules nanométriques déformables (dnvs) pour l'administration transmucosale et transdermique de médicaments à travers la barrière hémato-encéphalique
CN106006593B (zh) * 2016-05-18 2018-08-17 西南交通大学 一种简便高效的纳米磷酸钙制备方法
WO2017201528A1 (fr) 2016-05-20 2017-11-23 The University Of Chicago Nanoparticules pour chimiothérapie, thérapie ciblée, thérapie photodynamique, immunothérapie et n'importe quelle combinaison de ces dernières
JP7364552B2 (ja) 2017-08-02 2023-10-18 ザ ユニバーシティ オブ シカゴ Ｘ線誘起光線力学療法、放射線療法、放射線力学療法、化学療法、免疫療法、及びそれらの任意の組み合わせのための、ナノスケール金属有機層及び金属有機ナノプレート
CN112316153B (zh) * 2020-11-24 2023-08-25 中山大学附属第七医院(深圳) 一种抗肿瘤的药物组合物及其应用
CN112675130B (zh) * 2020-12-29 2023-04-25 中国人民解放军总医院第三医学中心 空心纳米颗粒在制备骨质疏松治疗药物中的应用
CN112724175A (zh) * 2020-12-30 2021-04-30 华南理工大学 一种米诺膦酸钙配合物及其制备方法
KR20240027761A (ko) 2021-06-28 2024-03-04 비온디스 비.브이. 인항원을 포함하는 접합체 및 요법에서의 이의 용도
PL441261A1 (pl) * 2022-05-24 2023-11-27 Politechnika Warszawska Formulacja nanocząstek fosforanu wapnia o zwiększonym wychwycie komórkowym modyfikowanych lecytyną jako nośnika bisfosfonianów oraz sposób jej wytwarzania

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DE10016559A1 (de) * 2000-04-03 2001-10-18 Eucro Europe Contract Res Gmbh System für den Transport von Aktivstoffen in einem biologischen System
FR2848850B1 (fr) * 2002-12-20 2005-06-24 Guerbet Sa Nouvelles compositions de particules magnetiques recouvertes de derives gem-bisphosphonates.
PE20070360A1 (es) * 2005-09-01 2007-04-19 Novartis Ag Composiciones de liposomas
US20070218116A1 (en) * 2006-03-14 2007-09-20 Schwendener Reto A Compositions and methods for the treatment of tumors and tumor metastases
US20100015068A1 (en) * 2006-07-06 2010-01-21 Massachusetts Institute Of Technology Methods and Compositions For Altering Biological Surfaces
WO2008008917A2 (fr) * 2006-07-12 2008-01-17 Mayo Foundation For Medical Education And Research Particules d'hydroxyapatite
US8821943B2 (en) * 2006-09-12 2014-09-02 Board Of Regents Of The University Of Nebraska Methods and compositions for targeted delivery of therapeutic agents
EP2255199A1 (fr) * 2008-02-19 2010-12-01 Jovesis Inc. Compositions de microparticules pour modifier des cellules favorisant le cancer
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DK2621539T3 (en) 2018-07-30
ITFI20100206A1 (it) 2012-04-02
ES2678498T3 (es) 2018-08-13
IT1401882B1 (it) 2013-08-28
PT2621539T (pt) 2018-07-27
US20140086979A1 (en) 2014-03-27
WO2012042024A1 (fr) 2012-04-05
ES2678498T8 (es) 2018-09-28
US9226897B2 (en) 2016-01-05

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